1. Field of the Invention
The present invention relates to a carboxylic acid derivative having a fused ring and a pharmacologically acceptable salt thereof. More specifically, it relates to a novel carboxylic acid derivative having a fused ring which exhibit agonism for retinoic acid receptors and a pharmacologically acceptable salt thereof.
2. Prior Art
Retinoic acid is a substance essential to the growth and life support of human being and other mammals. It has been known that retinoic acid acts as a morphogenetic factor in ontogenesis and functions variously in the differentiation and proliferation of cells of adults. For example, it has been known that the acid participates in cornification, formation of hair, functions of sebaceous gland and so on with respect to the epidermis, in metabolism of bone and cartilage with respect to the connective tissue, in regulation of immune functions with respect to the immune system, in differentiation of nerve cells with respect to the nervous system, in differentiation and proliferation of blood cells with respect to the hemic system, and in the lipid metabolism, the mineral metabolism and the basal metabolism and so on. These various physiological actions of retinoic acid are exhibited by various control mechanisms through retinoid receptor family present in the cell nucleus, for example, by regulating the expression of transcription activators, by regulating the expression of enzymes such as collagenase, tissue plasminogen activator or tyrosine kinase, or by regulating the production of cytokines such as IL-6.
The connections of the above physiological actions of retinoic acid with various diseases have recently been elucidated gradually, and in particular, differentiation-inducing therapy with all-trans retinoic acid has attracted attention as a new therapeutic method for some cancers such as acute promyelocytic leukemia.
With respect to retinoic acid, however, there have appeared problematic tolerance due to the induction of P450 which is a hepatic metabolic enzyme, adverse effects due to accumulation, and other problems. Under these circumstances there have been expected research and development of novel retinoid-related compounds which can be substituted for retionic acid as preventive and therapeutic drugs for various diseases.
Under the above circumstances, the inventors of the present invention have found that the desired objects can be attained by carboxylic acid derivatives having fused rings which will be described, and the present invention has been accomplished on the basis of this finding.
Namely, the present invention relates to a carboxylic acid derivative having a fused ring which is represented by the formula (A), or a pharmacologically acceptable salt thereof: 
{wherein the rings L and M are fused with each other; the symbol  represents a single bond or a double bond; X represents a group represented by the formula: xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94, or a group represented by the formula: 
(wherein R1 represents hydrogen, halogeno, optionally substituted lower alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted lower alkoxy, optionally substituted aryloxy, optionally substituted heteroaryloxy, optionally substituted cycloalkylalkyl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl, optionally substituted cycloalkyloxy, optionally substituted cycloalkylalkyloxy, optionally substituted arylalkyloxy, optionally substituted heteroarylalkyloxy, optionally substituted alkenyl or optionally substituted alkynyl), and x is an integer of 0 or 1;
Y represents a group represented by the formula: xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94, or a group represented by the formula: 
(wherein R2 represents hydrogen, halogeno, optionally substituted lower alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted lower alkoxy, optionally substituted aryloxy, optionally substituted heteroaryloxy, optionally substituted cycloalkylalkyl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl, optionally substituted cycloalkyloxy, optionally substituted cycloalkylalkyloxy, optionally substituted arylalkyloxy, optionally substituted heteroarylalkyloxy, optionally substituted alkenyl or optionally substituted alkynyl), and y is an integer of 0 or 1;
Z represents a group represented by the formula: xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94, or a group represented by the formula: 
(wherein R3 represents hydrogen, halogeno, optionally substituted lower alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted lower alkoxy, optionally substituted aryloxy, optionally substituted heteroaryloxy, optionally substituted cycloalkylalkyl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl, optionally substituted cycloalkyloxy, optionally substituted cycloalkylalkyloxy, optionally substituted arylalkyloxy, optionally substituted heteroarylalkyloxy, optionally substituted alkenyl or optionally substituted alkynyl), and z is an integer of 0 or 1;
P represents a group represented by the formula: xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94, or a group represented by the formula: 
(wherein R4 represents hydrogen, halogeno, optionally substituted lower alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted lower alkoxy, optionally substituted aryloxy, optionally substituted heteroaryloxy, optionally substituted cycloalkylalkyl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl, optionally substituted cycloalkyloxy, optionally substituted cycloalkylalkyloxy, optionally substituted arylalkyloxy, optionally substituted heteroarylalkyloxy, optionally substituted alkenyl or optionally substituted alkynyl), and p is an integer of 0 or 1;
Q represents a group represented by the formula: xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94, or a group represented by the formula: 
(wherein R5 represents hydrogen, halogeno, optionally substituted lower alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted lower alkoxy, optionally substituted aryloxy, optionally substituted heteroaryloxy, optionally substituted cycloalkylalkyl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl, optionally substituted cycloalkyloxy, optionally substituted cycloalkylalkyloxy, optionally substituted arylalkyloxy, optionally substituted heteroarylalkyloxy, optionally substituted alkenyl or optionally substituted alkynyl), and q is an integer of 0 or 1;
U represents a group represented by the formula: xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94, or a group represented by the formula: 
(wherein R6 represents hydrogen, halogeno, optionally substituted lower alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted lower alkoxy, optionally substituted aryloxy, optionally substituted heteroaryloxy, optionally substituted cycloalkylalkyl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl, optionally substituted cycloalkyloxy, optionally substituted cycloalkylalkyloxy, optionally substituted arylalkyloxy, optionally substituted heteroarylalkyloxy, optionally substituted alkenyl or optionally substituted alkynyl), and w is an integer of 0 or 1;
V represents a group represented by the formula: xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94, or a group represented by the formula: 
[wherein R7 represents hydrogen, halogeno, optionally substituted lower alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted lower alkoxy, optionally substituted aryloxy, optionally substituted heteroaryloxy, optionally substituted cycloalkylalkyl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl, optionally substituted cycloalkyloxy, optionally substituted cycloalkylalkyloxy, optionally substituted arylalkyloxy, optionally substituted heteroarylalkyloxy, optionally substituted alkenyl, optionally substituted alkynyl or a group represented by the formula: 
(wherein A and B each independently represent an optionally substituted aromatic hydrocarbon ring or an optionally substituted unsaturated heterocycle, and D represents optionally protected carboxyl)], and v is an integer of 0 or 1; and
W represents a group represented by the formula: xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94, or a group represented by the formula: 
[wherein R8 represents hydrogen, halogeno, optionally substituted lower alkyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted lower alkoxy, optionally substituted aryloxy, optionally substituted heteroaryloxy, optionally substituted cycloalkylalkyl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl, optionally substituted cycloalkyloxy, optionally substituted cycloalkylalkyloxy, optionally substituted arylalkyloxy, optionally substituted heteroarylalkyloxy, optionally substituted alkenyl, optionally substituted alkynyl or a group represented by the general formula: 
(wherein A and B each independently represent an optionally substituted aromatic hydrocarbon ring or an optionally substituted unsaturated heterocycle, and D represents optionally protected carboxyl)], and w is an integer of 0 or 1;
with the provisos that the symbol  in the formula: 
employed in the above definition of X, Y, Z, P, Q, U, V and W represents a single bond or a double bond, that two of R1, R2, R3, R4, R5, R6, R7 and R8 adjacent to each other together with the carbon atoms to which they are bonded respectively may form a ring which may contain a heteroatom or be substituted, that x, y, z and p must satisfy the relationship: 4xe2x89xa7x+y+z+pxe2x89xa73, and u, v, w and q must satisfy the relationship: 4xe2x89xa7u+v+w+qxe2x89xa73, that either of V and W is a group of the formula: 
(wherein Rk refers to R7 or R8), wherein R7 or R8 is a group represented by the formula: 
(wherein A and B each independently represent an optionally substituted aromatic hydrocarbon ring or an optionally substituted unsaturated heterocycle, and D represents optionally protected carboxyl), and that the compounds represented by the formula (A) wherein the ring L is completely saturated are excepted{.
Further, the present invention provides a medicament composition comprising a pharmacologically effective amount of the above carboxylic acid derivative having a fused ring or a pharmacologically acceptable salt thereof or a hydrate of the salt and a pharmacologically acceptable carrier.
Furthermore, the present invention provides a retinoic acid receptor agonist which is the above carboxylic acid derivative having a fused ring or a pharmacologically acceptable salt thereof or a hydrate of the salt.
The present invention also relates to a preventive and therapeutic agent for diseases against which retinoic acid receptor agonism is efficacious.
Additionally, the present invention provides a method for the prevention and treatment of diseases against which the retinoic acid receptor agonism is efficacious by administering a pharmacologically effective amount of the above carboxylic acid derivative having a fused ring or a pharmacologically acceptable salt thereof or a hydrate of the salt to a patient with such diseases, and use of the above carboxylic acid derivative having a fused ring or a pharmacologically acceptable salt thereof or a hydrate of the salt in preparing a remedy for diseases against which the retionic acid receptor agonism is efficacious.
In the above definition of the formula (A), the term xe2x80x9chalogensxe2x80x9d used in the definition of R1, R2, R3, R4, R5, R6, R7 and R8 refers to fluorine, chlorine, bromine or iodine.
The term xe2x80x9clower alkylxe2x80x9d used in the definition of R1, R2, R3, R4, R5, R6, R7 and R8 refers to a linear or branched alkyl group having 1 to 6 carbon atoms. Examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 2-ethylpropyl, n-hexyl, 1,2-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,1-diethylpropyl, 2,2-diethylpropyl, 1,2-diethylpropyl, 1-ethyl-2-methylpropyl, 1-methyl-2-ethylpropyl and 1,1- diethylethyl. These alkyl groups may be substituted with one to three halogen atoms such as fluorine, chlorine, bromine or iodine atoms. That is, the above linear or branched lower alkyl group includes also trifluoromethyl, dibromoethyl and so on.
The term xe2x80x9ccycloalkylxe2x80x9d used in the definition of R1, R2, R3, R4, R5, R6, R7 and R8 refers to one having 3 to 8 carbon atoms, and examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
The term xe2x80x9clower alkoxyxe2x80x9d used in the definition of R1, R2, R3, R4, R5, R6, R7 and R8 refers to a linear or branched alkoxy group having 1 to 6 carbon atoms. Examples thereof include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentyloxy, 1,2-dimethylpropyloxy, 1,1-dimethylpropyloxy, 2,2-dimethylpropyloxy, 2-ethylpropyloxy, n-hexyloxy, 1,2-dimethylbutyloxy, 2,3-dimethylbutyloxy, 1,3-dimethylbutyloxy, 1-ethyl-2-methylpropyloxy and 1-methyl-2-ethylpropyloxy. Further, these alkoxy groups may be substituted with one to three halogen atoms such as fluorine, chlorine, bromine or iodine atoms. That is, the above lower alkoxy group includes also trifluoromethoxy, dibromoethoxy and so on.
As defined above, R1, R2, R3, R4, R5, R6, R7 and R8 may be optionally substituted aryl, and the term xe2x80x9carylxe2x80x9d used in this case refers to phenyl, 1-naphthyl, 2-naphthyl, anthracenyl or the like.
As defined above, A and B may be each an optionally substituted aromatic hydrocarbon ring, and the term xe2x80x9caromatic hydrocarbon ringxe2x80x9d used in this case refers to benzene ring, naphthalene ring, anthracene ring or the like.
The term xe2x80x9coptionally substituted heteroarylxe2x80x9d used in the definition of R1, R2, R3, R4, R5, R6, R7 and R8 refers to a group derived from a monocyclic or fused ring containing one to four sulfur, oxygen or nitrogen atoms. Examples thereof include thienyl, furyl, benzothienyl, benzofuranyl, isobenzofuranyl, pyrrolyl, imidazolyl, pyrazolyl, isothiazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, indolyl, isoquinolinyl, quinolyl, phthalazinyl, quinoxalinyl, naphthyridinyl, quinazolinyl, acridinyl and furazanyl.
As defined above, A and B may be each an optionally substituted heterocycle, and the term xe2x80x9cheterocyclexe2x80x9d used in this case refers to a monocyclic or fused ring containing one to four sulfur, oxygen and/or nitrogen atoms. Examples thereof include thiophene ring, furan ring, benzothiophene ring, benzofuran ring, isobenzo- furan ring, pyrrole ring, imidazole ring, pyrazole ring, isothiazole ring, isoxazole ring, isoindole ring, indole ring, isoquinoline ring, quinoline ring, phthalazine ring, quinoxaline ring, naphthyridine ring, quinazoline ring, acridine ring and furazan ring.
As defined above, R1, R2, R3, R , R5, R6, R7 and R8may be each optionally substituted arylalkyl, and the term xe2x80x9carylxe2x80x9d used in this case refers to the same one defined above. Further, the term xe2x80x9calkylxe2x80x9d used in this case refers to the same one defined above with respect to the lower alkyl.
The term xe2x80x9coptionally substituted heteroarylalkylxe2x80x9d used in the definition of R1, R2, R3, R4, R5, R6, R7 and R8 refers to a group obtained by bonding the above heteroaryl group to any carbon atom of the above alkyl group.
The substituent constituting the above optionally substituted aryl, heteroaryl, arylalkyl or heteroarylalkyl group includes linear and branched lower alkyl groups such as methyl, ethyl, n-propyl and isopropyl; linear and branched lower alkoxy groups such as methoxy, ethoxy, n-propoxy and isopropoxy; halogeno groups such as fluorine, chlorine, bromine and iodine; optionally substituted aryl groups; optionally substituted heteroaryl groups; optionally substituted arylalkyl groups; optionally substituted heteroarylalkyl groups; halogeno groups; hydroxy; hydroxyalkyl groups; alkoxyalkyl groups; and so on.
As described above, D is optionally protected carboxyl, and examples of the protecting group for this carboxyl group include lower alkyl groups such as methyl, ethyl and t-butyl; optionally substituted phenylated lower alkyl groups such as p-methoxybenzyl, p-nitrobenzyl, 3,4-dimethoxybenzyl, diphenylmethyl, trityl and phenethyl; halogenated lower alkyl groups such as 2,2,2-trichloroethyl and 2-iodoethyl; lower alkanoyloxy lower alkyl groups such as pivaloyloxymethyl, acetoxymethyl, propionyloxymethyl, butyryloxymethyl, valeryloxymethyl, 1-acetoxyethyl, 2-acetoxyethyl, 1-pivaloyloxyethyl and 2-pivaloyloxyethyl; higher alkanoyloxy lower alkyl groups such as palmitoyloxyethyl, heptadecanoyloxymethyl and 1-palmitoyloxyethyl; lower alkoxycarbonyloxy lower alkyl groups such as methoxycarbonyloxymethyl, 1-butoxycarbonyloxyethyl and 1-(isopropoxycarbonyloxy)ethyl; carboxylated lower alkyl groups such as carboxymethyl and 2-carboxyethyl; heteroaryl groups such as 3-phthalidyl; optionally substituted benzoyloxy lower alkyl groups such as 4-glycyloxybenzoyloxymethyl; (substituted dioxolene) lower alkyl groups such as (5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl; cycloalkylated lower alkanoyloxy lower alkyl groups such as 1-cyclohexylacetyloxyethyl; cycloalkyloxycarbonyloxy lower alkyl groups such as 1-cyclohexyloxycarbonyloxyethyl; and optionally substituted amino groups. That is, the term xe2x80x9coptionally protected carboxylxe2x80x9d refers to carboxyl or a group which can be cleaved either by chemical means or in vivo to give a carboxylic acid.
Examples of the pharmacologically acceptable salt according to the present invention include inorganic salts such as hydrochlorides, hydrobromides, sulfates and phosphates; organic acid salts such as acetates, maleates, tartrates, methanesulfonates, benzenesulfonates and toluenesulfonates; and amino acid salts such as aspartates and glutamates.
When the compounds according to the present invention are present as optical isomers, the present invention includes also such optical isomers.
The compounds according to the present invention can readily be prepared by conventional processes or combinations of two or more of them. An example of the preparation process will now be described.
Preparation Process 1
Compounds represented by the formula (A) wherein A is a pyrrole ring can be prepared by the following process. 
In this step, an allyl alcohol (2) is prepared by reacting an aldehyde (1) with an organometallic reagent in a conventional manner.
The organometallic reagent includes Grignard reagents, organolithium reagents, organozinc reagents, organocopper complexes and soon. Although any solvent inert to the reaction may be used in this step, the use of an etheric solvent such as ether or tetrahydrofuran is preferable. The reaction temperature may range from xe2x88x9278xc2x0 C. to the boiling point of the solvent, preferably from about xe2x88x9278xc2x0 C. to 20xc2x0 C.
(Step 2)
In this step, the allyl alcohol (2) prepared in the step (1) is oxidized into a vinyl ketone (3) in a conventional manner.
Although the oxidation may be conducted by any conventional process, the use of a suitable oxidizing agent is preferable. Examples of the oxidizing agent include activated manganese dioxide, pyridinium chlorochromate, pyridinium dichromate, Dess-Martin reagent and Swern oxidation reagent. Although any organic solvent inert to the reaction may be used for the oxidation, the use of dichloromethane, chloroform or acetone is preferable. The reaction temperature may range from about xe2x88x9278xc2x0 C. to the boiling point of the solvent, preferably from about xe2x88x9278xc2x0 C. to 20xc2x0 C.
(Step 3)
In this step, a diketone represented by the formula (5) is prepared from the vinyl ketone (3) prepared in the step (2) and an aldehyde (4) according to the process of Stetter et al. described in Org. Synth. 65, 26.
In this step, better results can be attained by using a thiazolium salt catalyst. In such a case, it is preferable to use triethylamine, sodium acetate or the like as the base. Further, the solvent to be used in the above reaction may be methanol, ethanol, N,N-dimethylformamide or the like. The reaction temperature is preferably about 60xc2x0 C. to the boiling point of the solvent.
(Step 4) In this step, the diketone (5) prepared in the step 3 is converted into a pyrrole represented by the formula (6) through a conventional treatment.
The objective compound (6) can be prepared by, for example, reacting the diketone (5) with an ammonium salt such as ammonium acetate or a primary amine. In this case, an alcoholic solvent such as methanol or ethanol or acetic acid may be used as the solvent. The reaction temperature is preferably about 70xc2x0 C. to the boiling point of the solvent.
(Step 5)
In this step, the pyrrole (6) prepared in the step 4 is conventionally hydrolyzed into a final objective compound represented by the formula (7). In this step, better results can be attained by using a base, particularly an aqueous solution of lithium hydroxide, sodium hydroxide, potassium hydroxide or the like. Preferable examples of the solvent to be used in this hydrolysis include alcohols such as methanol and ethanol and ethers such as tetrahydrofuran. The reaction temperature is preferably about 20xc2x0 C. to the boiling point of the solvent.
Next, another process is described with respect to the preparation of the diketone (5) used in the above Preparation process 1.
Preparation Process 1xe2x80x2
The diketone (5) can be prepared also by reacting the vinyl ketone (8) prepared in a similar manner to that of Preparation process 1 with the aldehyde (1) in the presence of a thiazolium salt catalyst according to the process of Stetter et al. In this process, better results can be attained by using as the base triethylamine, sodium acetate or the like. The solvent to be used in this process includes alcohols such as methanol and ethanol, N,N-dimethylformamide and so on. The reaction temperature is preferably about 60xc2x0 C. to the boiling point of the solvent.
Pharmacological Experimental Examples will now be described to illustrate the effects of the present invention.
Receptor Binding Assay Using Nuclear Extract Fraction of Cells Hearing RAR Genes Transferred Thereinto
Human RAR xcex1, xcex2 and xcex3 genes were transferred into BHK (Baby Hamster Kidney) cells to prepare cells constantly expressing RAR xcex1, xcex2 and xcex3 proteins. An experimental system for measuring the specific binding of all-trans retinoic acid for RARs was constructed by the use of a nuclear extract fraction of the cells, and the abilities of each compound to bind RARs were determined by measuring the inhibition against the specific binding. Further, the selectivity of each compound among RARs was determined by comparing the abilities of the compound to bind RARs with each other.
(1) Experimental Method
a) Preparation of Nuclear Extract Fraction
The above BHK cells (5xc3x97108) into which RAR genes had been transferred were suspended in 15 ml of solution A (sodium phosphate (pH7.4): 5 mM, monothioglycerol:10 mM, glycerol: 10% (v/v), phenylmethylsulfonyl fluoride (PMSF): 1 mM, aprotinin: 10 xcexcg/ml, and leupeptin: 25 xcexcg/ml). The resulting suspension was homogenized and centrifuged to remove the resulting supernatant. The resulting sediment was suspended in 15 ml of solution B (Tris-HCl (pH8.5): 10 mM, monothioglycerol: 10 mM, glycerol: 10% (v/v), PMSF: 1 mM, aprotinin: 10 xcexcg/ml, leupeptin: 25 xcexcg/ml, and KCl: 0.4 M). The resulting suspension was allowed to stand at 4xc2x0 C. for one hour, and subjected to ultracentrifugation under the conditions of 100,000xc3x97g, 4xc2x0 C. and one hour. The resulting supernatant was stored as the nuclear extract fraction in a frozen state at xe2x88x9280xc2x0 C. until the use (METHODS IN ENZYMOLOGY, 189, 248).
b) Receptor Binding Assay
180 xcexcl of the above fraction and 10 xcexcl of a dilution of all-trans retinoic acid or a test compound were added to each well of a 96-well plate made of polypropylene, followed by the addition of 10 xcexcl of 10 nM 3H-all-trans retinoic acid. The resulting plate was allowed to stand at 4xc2x0 C. for 16 hours. A solution containing 3% of charcoal and 0.3% of dextran was added to the resulting reaction mixture. The resulting mixture was centrifuged to remove free 3H-all-trans retinoic acid. The radioactivity of the resulting supernatant was determined by the use of a scintillation counter. The specific binding for each RAR was determined by assuming the radioactivity found when 500 times as much all-trans retionic acid was added to be the non-specific binding and subtracting it from the radioactivity determined above. The compounds which will be described below inhibited the binding of 3H-all-trans retinoic acid dependently on the concentration.
(2) Experimental Results
The concentration at which the binding of 3H-all-trans acid for each receptor is inhibited by 50%, i.e., IC50 was calculated from the specific binding for the RAR, and the activities are given in Table 1, which were calculated from the IC50 value of all-trans retinoic acid to be 1.
Measurement of the Activities of Accelerating Trans-cription Through RARs
Human RAR Expression Vectors and secretory alkaline phosphatase (PLAP) gene vectors (PLAP vectors) containing in a state integrated into the upstream a competent sequence whose expression is inhibited through binding with RAR depending on a ligand were temporarily transferred into COS-1 (African green monkey kidney cells), and the PLAP which had been produced depending on a ligand and secreted into a culture medium was analyzed by the chemiluminescence method to determine the transcription-accelerating activity of each compound. Further, the selectivity of each compound among RARs was determined by comparing the transcription accelerating activities of the compound for the receptors with each other.
(1) Experimental Method
On a 60-mm culture dish were scattered 2.5xc3x97104 COS-1 cells. Four days after, human RAR xcex1, xcex2 and xcex3 expression vectors and PLAP vectors were transferred into the cells each in an amount of 4 xcexcg by the lipofection method. Another day after, the resulting cells were recovered, and put on a 96-well culture plate in an amount of 2xc3x97104 per unit well. Four hours after, the cells were put on a medium containing charcoal-treated fetal bovine serum, followed by the addition of a dilution of all-trans retinoic acid or a test compound. After the lapse of 36 hours, the supernatant was recovered and the resulting samples were treated at 65xc2x0 C. for 10 minutes to eliminate the non-specific activity. 15 xcexcl of each sample was mixed with 60 xcexcl of a 28 mM sodium carbonate buffer(pH10), followed by the addition of 75 xcexcl of Smilight (trade name, a product of Sumitomo Metal Industries, Ltd., substrate for chemiluminescence). The resulting mixture was reacted at 37xc2x0 C. for 30 minutes and the intensity of luminescence was determined. The compounds which will be described below induced the transcription activities of RARs dependently on the concentration.
(2) Experimental Results
With the transcription activity induced by 1 xcexcM all-trans retinoic acid being assumed to be 100%, the concentration at which 30% of the activity is exhibited, i.e., ED30 was calculated for each compound. The relative activities of the compounds for each receptor are given in Table 2, which were calculated by assuming the ED30 value of all-trans retinoic acid to be 1.
The above Pharmacological Experimental Examples have revealed that the carboxylic acid derivatives represented by the formula (A) or pharmacologically acceptable salts thereof exhibit retinoic acid receptor agonism. Accordingly, the derivatives according to the present invention are useful as preventive and therapeutic agents for diseases against which the retinoic acid receptor agonism is efficacious. That is, the derivatives are usable as preventive and therapeutic agents for various cornification anomalies and skin diseases such as xeroderma pigmentosum, psoriasis, arthropathia psoriatica, acne or leukoplakia; various alopeciae such as alopecia areata, seborrheic alopecia or cachectic alopecia; various osteoporoses and osteopeniae such as postmenopausal osteoporosis, senile osteoporosis, steroidal osteoporosis, idiopathic osteoporosis, diabetic osteopenia, rheumatoid osteopenia or renal osteomalacia; diseases of bone and joint such as ectopic hyperostosis, osteoarthritis or shoulder periarthritis; autoimmune diseases such as chronic rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, Behcet""s disease, mycosis fungoides, systemic scleroderma, sudden thrombo-cytopenic purpura, myasthenia gravis, dermatomyositis or nodular arteriosclerosis; various leukemiae such as acute promyelocytic leukemia, acute myelocytic leukemia or chronic leukemia; rejections of graft in organ transplantation; graft versus host diseases (GVHD) in born marrow transplantation or stem cell transplantation; nephropathies such as nephrotic syndrome; glomerulonephritis; malignant lymphomas such as mycosis fungoides; squamous cell carcinomas such as squamous cell carcinoma of head and neck; solid carcinomas such as bladder cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, prostatic cancer or pancreatic cancer; inflammations and allergic diseases such as atopic dermatitis or asthma; immune deficiencies and intractable infections such as immunodeficiency diseases, infections with cytomegalovirus due to lowered immune function or of fetus or opportunistic infection; hyperthyroidism; hypercalcemia; various fibroses such as pulmonary fibrosis, hepatic fibrosis or hepatic cirrhosis; atherosclerosis and restenosis after reconstructive operation of blood circulation; other nonmalignant hyperplastic diseases such as endometrial hyperplasia, benign prostatic hypertrophy, proliferative vitreoretinopathy and dysplasia; diseases related to metabolism and transport of lipid such as hyperlipidemia; diabetes; wounds; dry eye syndrome; or solar skin injury; and as apoptosis induction accelerators.
The compounds of the present invention are lowly toxic and highly safe, being useful also in this respect.
When the compounds of the present invention are to be administered for the above diseases, the route of administration may suitably be selected. Specifically, they may be orally administered as preventive or therapeutic agents in the form of tablets, powders, granules, capsules, syrups or the like, or may be parenterally administered in the form of suppositories, injections, external preparations or drops.
Although the dosage of the compound remarkably depends on the kind of diseases, the extent of symptom, the interval from sideration to the first administration, the age, sex and sensitivity of patient or the like, the compound may be administered generally in a dosage of about 0.03 to 1000 mg, preferably 0.1 to 500 mg, still preferably of 0.1 to 100 mg per adult a day in several portions.
When the compound is to be administered as an injection, the dosage of the compound is generally about 1 to 3000 xcexcg/kg, preferably about 3 to 1000 xcexcg/kg.
The compounds of the present invention may be formulated into pharmaceutical preparations by the use of conventional preparation carriers according to conventional processes.
Specifically, a solid pharmaceutical preparation for oral administration according to the present invention can be formulated by adding a filler, binder, disintegrator, lubricant, colorant, corrigent, antioxidant and so on to a principal agent, and shaping the obtained mixture into tablets, coated tablets, granules, powders, capsules or the like according to conventional processes.
Examples of the filler include lactose, corn starch, sucrose, glucose, sorbitol, crystalline cellulose and silicon dioxide.
Examples of the binder include polyvinyl alcohol, polyvinyl ether, ethylcellulose, methylcellulose, acacia, tragacanth, gelatin, shellac, hydroxypropylcellulose, hydroxypropylmethylcellulose, calcium citrate, dextrin and pectin, and those of the lubricant include magnesium stearate, talc, polyethylene glycol, silica and hardened vegetable oils.
The colorant includes those authorized as pharmaceutical additives. The corrigent includes cocoa powder, menthol, aromatic powder, mentha oil, borneol, powdered cinnamic bark and so on. The antioxidant includes those authorized as pharmaceutical additives, for example, ascorbic acid and xcex1-tocopherol. Of course, the tablets and granules may be coated with sugar, gelatin or the like at need.
An injection according to the present invention can be formulated by a conventional process which comprises adding a pH regulator, buffer, suspending agent, solubilizing agent, stabilizer, tonicity agent, antioxidant and/or preservative to a principal agent at need and, if necessary, freeze-drying the resulting mixture. Such an injection may be administered intravenously, subcutaneously or intramuscularly.
Examples of the suspending agent include methylcellulose, polysorbate 80, hydroxyethylcellulose, acacia, tragacanth powder, carboxymethylcellulose sodium and polyoxyethylene sorbitan monolaurate.
The solubilizing agent includes polyoxyethylene hardened castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate and so on.
Examples of the stabilizer include sodium sulfite, sodium metasulfite and ether. Examples of the preservative include methyl p-hydroxybezoate, ethyl p-hydroxybenzoate, sorbic acid, phenol, cresol and chlorocresol.